Siderophore production

Qualitative and quantitative estimation of siderophore production was done by CAS assay (Schwyn and Neilands 1987). Specific tests were carried out for the identification of hydroxamate and catecholate types of siderophores following the standard methods (Arnow 1937). For qualitative estimation, chrome azurol S solution was prepared and added to melted King’s B agar medium in the ratio 1:15. Spot inoculation at the centre of the CAS plate was done from actively growing cultures of Pseudomonas. Colonies exhibiting an orange halo after 3 days incubation (28 ± 2 °C) were considered positive for siderophore production and the diameter of the orange halo was measured. Simultaneously succinate medium (broth) was also used for qualitative estimation of siderophore production on the basis of fluorescence observed after 3 days incubation (28 ± 2 °C).

For siderophore quantification, actively growing cultures of Pseudomonas was inoculated to 20 mL King’s B broth in 100 mL flasks and incubated for 3 days at 28 ± 2 °C. The bacterial cells were removed by centrifugation at 3000 rpm for 5 min. 0.5 mL of the culture supernatant was then mixed with 0.5 mL of CAS solution and 10 µl shuttling reagent (sulfosalicyclic acid). After 20 min of incubation, the absorbance of colour obtained was determined using spectrophotometer at 630 nm. Un-inoculated King’s B broth was used as blank while reference solution was prepared by adding CAS dye and shuttle solution to King’s B and absorbance was recorded. Values of siderophore released in King’s B was expressed as percent siderophore units and calculated using the formula: (A _r − A _s)/A _r × 100; where A _r is the absorbance of reference solution and A _s is the absorbance of samples.

Isolates were inoculated on King’s B medium supplemented with a strong iron chelater 8- Hydroxyquinoline (50 mg/L) (De Brito et al. 1995) and incubated at 28 ± 2 °C for 48–72 h. Only those bacteria that produce a more avid iron chelator will grow.

Arnow’s assay was used for qualitative determination of catechol type of siderophore. Actively growing cultures of Pseudomonas were inoculated to 20 mL King’s B broth in 50 mL tubes and incubated for 3 days at 28 ± 2 °C. The bacterial cells were removed by centrifugation at 3000 rpm for 5 min. Three milliliter of the culture supernatant was then mixed with 0.3 mL of 5 N HCl solution, 1.5 mL of Arnow’s reagent (10 g NaNO₂, 10 g Na₂MoO₄·2H₂O dissolved in 50 mL distilled water) and 0.3 mL of 10 N NaOH. After 10 min the presence or absence of pink colour was observed and noted.

This test is based on the capacity of hydroxamic acid to reduce tetrazolium salt by hydrolysis of hydroxymate groups using a strong alkali. The reduction and release of alkali shows red colour to a pinch of tetrazolium salt when 1–2 drops of 2 N NaOH and 0.1 mL of test sample is added. Instant appearance of a deep red colour indicated the presence of hydroxamate siderophore.

One milliliter of the culture supernatant was mixed with freshly prepared 0.5 mL of 2% aqueous FeCl₃ and observed for the presence and absence of deep red colour.